The overall objective of this project is to define futher the roles of cuproproteins in iron metabolism. We propose to continue our studies of the role of ceruloplasmin (Cp) in the movement of iron from hepatocytes, reticuloendothelial cells and mucosal cells to plasma. Previous studies have shown that iron flow is restricted in the hypoceruloplasminemia of copper deficiency in swine and rats, and normal outflow is restored when Cp is administered. We plan to determine the cell or cells acted on by Cp by specifically labelling hepatocytes with 59Fe-Hemoglobin-haptoglobin, labelling reticuloendothelial cells with 55Fe-Damaged red cells, and measuring outflow of these isotopes when Cp is given. The effect of Cp on mucosal cells will be determined by measuring iron absorption in an isolated gut loop perfused with blood containing various amounts of Cp. The mechanism of action of Cp will be studied in suspensions of hepatocytes or macrophages. Evidence that Cp (1) interacts with a membrane site, (2) donates copper to an intracellular protein or (3) acts only by its ferroxidase activity will be studied. We also plan to study the movement of iron into mitochondria and the nature and function of the mitochondrial iron pool. Using isolated mitochondria exposed to transferrin-59Fe, we plan to evaluate the hypothesis that transferrin enters normoblasts and gives up its iron directly to mitochondria. Mitochondria will be loaded with excess iron by in vivo (vitamin B6 deficiency) or in vitro techniques, and the matrix iron fraction collected and purified. Its function as an iron storage compound and/or a substrate for heme synthesis will be evaluated. The role of superoxide dismutase (SOD) in protecting the erythrocyte from free radical attack will be studied. Previous in vitro work suggests that superoxide (O2) causes methemoglobin formation and that this can be prevented with SOD. SOD deficiency will be induced by copper deprivation and phenylhydrazine given. Effects on methemoglobin and Heinz-body formation will be measured.